The background of the present invention is twofold in that it relates to: (1) biological organisms which have been genetically transformed; and (2) a study of genetic material related to amyloidosis.
Both of these areas are discussed below.
2.1. Genetic Transformations
For sometime it has been known that it is possible to carry out the genetic transformation of a zygote (and the embryo and mature organism which result therefrom) by the placing or insertion of exogenous genetic material into the nucleus of the zygote or to any nucleic genetic material which ultimately forms a part of the nucleus of the zygote. The genotype of the zygote and the organism which results from a zygote will include the genotype of the exogenous genetic material. Additionally, the inclusion of exogenous genetic material in the zygote will result in a phenotype expression of the exogenous genetic material.
The genotype of the exogenous genetic material is expressed upon the cellular division of the zygote. However, the phenotype expression, e.g., the production of a protein product or products of the exogenous genetic material, or alterations of the zygote's or organism's natural phenotype, will occur at that point of the zygote's or organism's development during which the particular exogenous genetic material is active. Alterations of the expression of the phenotype include an enhancement or diminution in the expression of a phenotype or an alteration in the promotion and/or control of a phenotype, including the addition of a new promoter and/or controller or supplementation of an existing promoter and/or controller of the phenotype.
The genetic transformation of various types of organisms is disclosed and described in detail in U.S. Pat. No. 4,873,191, issued Oct. 10, 1989, which is incorporated herein by reference to disclose methods of producing transgenic organisms. The genetic transformation of organisms can be used as an in vivo analysis of gene expression during differentiation and in the elimination or diminution of genetic diseases.
The genetic transformation of a zygote (and the organisms which matures therefrom) is carried out by the addition of exogenous genetic material in a manner such that the exogenous genetic material becomes part of the nucleic portion of the zygote prior to a division of the zygote. If the exogenous genetic material is added after mitosis or cell division of the zygote, the exogenous genetic material must be added to each resulting nucleus. However, there is a possibility that the exogenous genetic material may not be integrated into and become a part of the genetic material of the zygote and the organism which results therefrom. Thus, the exogenous genetic material can be added to any nucleic genetic material which ultimately forms a part of the nucleus of the zygote, including the zygote nucleus.
The nucleic genetic material of the organism being transformed must be in a physical state which enables it to take up the exogenous genetic material. There are numerous ways of accomplishing this. For example, the exogenous genetic material can be placed in the nucleus of a primordial germ cell which is diploid, e.g., a spermatogonium or oogonium. The primordial germ cell is then allowed to mature to a gamete, which is then united with another gamete or source of a haploid set of chromosomes to form a zygote.
The exogenous genetic material can be placed in the nucleus of a mature egg. It is preferred that the egg be in a fertilized or activated (by parthenogenesis) state. After the addition of the exogenous genetic material, a complementary haploid set of chromosomes (e.g., a sperm cell or polar body) is added to enable the formation of a zygote. The zygote is allowed to develop into an organism such as by implanting it in a pseudopregnant female. The resulting organism is analyzed for the integration of the exogenous genetic material. If positive integration is determined, the organism can be used for the in vivo analysis of the gene expression, which expression is believed to be related to a particular genetic disease.
Attempts have been made to study a number of different types of genetic diseases utilizing such transgenic animals. Attempts related to studying Alzheimer's disease are disclosed within published PCT application WO 89/06689 and PCT application WO 89/06693, both published on Jul. 27, 1989, which published applications are incorporated herein by reference to disclose genetic sequences coding for Alzheimer's .beta.-amyloid protein and the incorporation of such sequences into the genome of transgenic animals.
As described in detail below, the production of .beta.-amyloid protein is believed to be related to Alzheimer's disease. However, a serious obstacle to elucidating the molecular mechanism involved in amyloid synthesis and deposition in an Alzheimer's diseased brain has been the unavailability of convincing animal models for this uniquely human disorder. Published PCT applications WO 89/06689 and WO 89/06693 disclose particular DNA sequences believed to be related to the production of amyloid. These particular sequences are fused to newly developed tumor virus vectors, derived from Simian Virus 40 (SV 40) and the JC virus to produce constructs. These constructs are utilized to transfect cells and transgenic mice to establish models for amyloid overexpression, which may be related to amyloid accumulation in the Alzheimer's-diseased brain.
The transgenic animals produced in accordance with the present invention are intended to provide an experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer's disease and to serve as tools for screening drugs that may have potential application as therapeutic agents to prevent or limit amyloid accumulation.
2.2. Alzheimer's Disease and .beta.-Amyloid Protein
It is estimated that over 5% of the U.S. population over 65 and over 15% of the U.S. population over 85 are beset with some form of Alzheimer's disease (Cross, A. J., Eur. J. Pharmacol. (1982) 82:77-80; Terry, R. D., et al., Ann. Neurol. (1983) 14:497-506). It is believed that the principal cause for confinement of the elderly in long term care facilities is due to this disease, and approximately 65% of those dying in skilled nursing facilities suffer from it.
Certain facts about the biochemical and metabolic phenomena associated with the presence of Alzheimer's disease are known. Two morphological and histopathological changes noted in Alzheimer's disease brains are neurofibrillary tangles (NFT) and amyloid deposits. Intraneuronal neurofibrillary tangles are present in other degenerative diseases as well, but the presence of amyloid deposits both in the interneuronal spaces (neuritic plaques) and in the surrounding microvasculature (vascular plaques) seems to be characteristic of Alzheimer's. Of these, the neuritic plaques seem to be the most prevalent (Price, D. L., et al., Drug Development Research (1985) 5:59-68). Plaques are also seen in the brains of aged Down's Syndrome patients who develop Alzheimer's disease.
The protein which makes up the bulk of these plaques has been partially purified and sequenced. Plaque-rich brains of deceased Alzheimer's patients have been used as a source to extract an approximately 4.2 kd "core" polypeptide, amyloid plaque core protein (APCP), herein referred to as ".beta.-amyloid core protein." This peptide was designated .beta.-protein by Glenner, G., et al. (Biochem Biophys Res Commun (1984) 120:885-890). The amino acid sequence of the amino-terminus has been determined Glenner, G., et al., Biochem. Biophys. Res. Commun. (1984) 122:1131-1135; Masters, C. L., et al., Proc Natl. Acad. Sci. (USA) (1985) 82:4245-4259! and the amino acid sequences reported by the two groups are identical except that Glenner et al., report a glutamine at position 11 for Alzheimer's disease cerebral vascular amyloid whereas Masters et al., report glutamic acid at position 11. Also, the former authors report that the cerebral vascular amyloid has a unique amino-terminus while the latter authors report that the form found in amyloid plaque cores has a "ragged" amino-terminus i.e., peptides isolated from this source appear to be missing 3, 7, or 8 amino acids from the amino-terminus. Both groups have shown that the same peptide is found in the amyloid plaque cores and vascular amyloid of adult Down's syndrome-afflicted individuals and report glutamic acid at position 11.
Further studies on the .beta.-amyloid core protein were also conducted by Roher, A., et al., Proc. Natl. Acad. Sci. (USA) (1986) 83:2662-2666 which showed the complete amino acid composition of the .beta.-protein, and verified that it matched that of no known protein. The compositions obtained were, however, evidently not in agreement with those of Allsop, D., et al., Brain Res (1983) 259:348.gtoreq.352; nor were they in agreement with those published by Glenner or Masters (supra).
Wong, C. W., et al., Proc. Natl. Acad. Sci. (USA) (1985) 82:8729-8732 showed that a synthetic peptide which was homologous to the first ten amino acids of the .beta.-amyloid core protein described by Masters (supra) was able to raise antibodies in mice and that these antibodies could be used to stain not only amyloid-laden cerebral vessels, but neuritic plaques as well. These results were confirmed by Allsop, D. et al., Neuroscience Letters (1986) 68:252-256 using monoclonal antibodies directed against a synthetic peptide corresponding to amino acids 8-17. Thus, in general, the plaque protein found in various locations of the brain of Alzheimer's patients appears to be similar in immunoreactivity. It is highly insoluble, as shown by the inability to achieve solubilization in many commonly used denaturants such as detergents and chaotropic agents (Masters (supra), Allsop, D., et al. (supra)).
It is believed, by analogy to some other amyloid proteins, that .beta.-amyloid core protein may be formed from a precursor in the peripheral circulatory system or lymphatic system. There are six known instances of disease-associated amyloid deposits in which the nature of the precursor protein for the amyloid protein is known: for primary amyloidosis, the source is an immunoglobulin light chain; for secondary amyloidosis, the precursor is amyloid A protein; for familial amyloid polyneuropathy and senile cardiac amyloidosis, prealbumin also known as transthyreitin or a variant thereof; for medullary carcinoma of thyroid, a procalcitonin fragment; and for hereditary cerebral hemorrhage, gamma-trace fragment which has been shown to be cystatin C. (See, e.g., Glenner, G. New. Eng. J. Med. (1980) 302:1283; Sletton, K., et al., Biochem. J. (1981) 195:561; Benditt, et al., FEBS. Lett. (1971) 19:169; Sletton, K., et al., Eur. J. Biochem. (1974) 41:117; and Sletton, K., et al., J. Exp. Med. (1976) 143:993). The foregoing is a partial list and there are at least a number of additional references with regard to procalcitonin fragment as a precursor for the amyloid of the thyroid carcinoma. Alternatively, or additionally, such a precursor for .beta.-amyloid core protein may be produced in the brain or elsewhere and is specifically deposited in the brain.
It has been described that a protein containing the .beta.-amyloid core protein (referred to as A4) sequence within the framework of a larger protein exists (Kang, J., et al., Nature (1987) 325:733-736). This protein, which is a potential precursor in vivo to the .beta.-amyloid core protein, was predicted from the sequence of a cDNA clone isolated from a human fetal brain tissue cDNA library and consists of 695 amino acid residues (referred to as A695) wherein the amino terminus of the .beta.-amyloid core protein begins at position 597. By analogy to the above described series, it may be that such a precursor or a fragment thereof circulates in the serum at a higher level differentiable in Alzheimer's victims relative to unafflicted individuals. Alternatively or additionally, such differences may be detected in the cerebral spinal fluid.
It appears as though there are a number of precursor proteins in addition to A695, which was described by Kang et al. One such precursor protein is described in copending U.S. application Ser. No. 361,912, filed Jun. 6, 1989, by researchers from the same research organization as the present inventors (A751). Others have characterized an additional amyloid precursor protein (see Kitaguchi et al., Nature (1988) 331:530-532, which is slightly larger, 770 amino acids. It is pointed out that these A751 and A770 proteins contain an approximately 57 amino acid insert beyond A695. This particular 57 amino acid insert sequence is highly homologous to a number of Kunitz-type inhibitors which are specific for a number of serine proteases. An additional 19 amino acids are present adjacent to the 57 amino acid insert in the A770 form.
As indicated by the above publications and numerous other publications not cited, the genetic material encoding for the production of .beta.-amyloid precursor proteins are the subject of intensive study. However, at present, there is no direct verifiable information available on the specific mechanisms that regulate the production and deposit of amyloid protein in an Alzheimer's diseased brain. It is known that the genetic material encoding for the production of .beta.-amyloid precursor protein is on chromosome 21. Further, numerous studies suggest that there are complex interactions involving the genetic material on this chromosome. Such interactions are believed to involve the production of precursor proteins, proteases and protease inhibitors. Further, it is believed that in individuals suffering from Alzheimer's disease these interactions are somehow skewed so that an unusually high content of .beta.-amyloid core protein is produced and/or deposited in the brain. The high .beta.-amyloid core protein concentration could be the result of a variety of biochemical activities including the overproduction of such proteins and/or the inability to cleave sufficient numbers of such proteins once cleaved.
The transgenic mammals of the present invention will provide insights with respect to how and where these interactions occur and thus provide more useful models for testing the efficacy of certain drugs in preventing or reducing the accumulation of .beta.-amyloid core protein in the brain. The transgenic non-human mammals of the present invention include recombinant genetic material comprised of specific segments of .beta.-amyloid precursor proteins which segments are fused to specific promoters capable of expressing the protein in specific tissues such as nerve tissues generally and/or specific types of nerve tissue, e.g., the brain.